Preparation method for biocompatible membrane for biosensor, and glucose sensor

Abstract

A preparation method for a biocompatible membrane for a biosensor, and a glucose sensor. The preparation method specifically comprises: sequentially performing a main-chain monomer polymerization reaction, a side-chain modification reaction, a side-chain coenzyme modification reaction, and coating to form a biocompatible membrane. The biocompatible membrane can be used in a biosensor, not only enabling precise regulation of chemical substance permeation and expanding the linear range of detection of the biosensor for chemical substances, but also providing a cofactor for the biosensor, thereby enhancing the stability and durability of the biosensor.

Description

Preparation method of biocompatible membrane for biosensor and glucose sensor TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to a preparation method of biocompatible membrane for biosensor and glucose sensor. BACKGROUND

[0002] An electrochemical biosensor is an analytical device based on biological substances and their derivative materials or biomimetic materials, which can combine technologies such as electrochemistry, polymer chemistry, biochemistry and electronic circuits, and usually contains biological enzymes that can specifically recognize target substances. The biochemical reaction signal is converted into an electrical signal for detection, which is used for quantitative detection of chemical substances in the human body, such as glucose, uric acid, etc.

[0003] In an implantable continuous blood glucose detection sensor, the sensing layer of the working electrode directly contacts the chemical substance, and the enzyme on the electrode reacts with the target glucose and exchanges electrons with the electrode to convert the chemical reaction signal into an electrical signal. There is a certain linear relationship between the electrical signal and the concentration of the chemical substance, and the concentration of the chemical substance can be determined according to the strength of the electrical signal. In the enzyme electrode direct electron transfer blood glucose reaction system, the cofactor (coenzyme factor) contained in the sensing layer plays a key role, but the cofactor is an exogenous small molecule that is lost during multiple uses of the sensor, resulting in insufficient amount of cofactor and affecting the accuracy of detection. In addition, when the sensing layer of the working electrode directly contacts the normal blood glucose concentration of the human body, the enzyme electrochemical kinetics is no longer limited by the glucose concentration, and the current value collected does not have a corresponding relationship with the chemical substance concentration. Therefore, in the direct electron transfer blood glucose biosensor, how to stabilize the cofactor related to the biological enzyme and effectively limit the glucose concentration to enable stable detection for multiple long times is a technical problem that needs to be solved by those skilled in the art.

[0004] SUMMARY

[0005] In view of the problems of loss of cofactor during multiple uses of the existing biosensor and influence of high concentration of chemical substance on the accuracy of sensor detection, the present application proposes a preparation method of biocompatible membrane for biosensor and glucose sensor to overcome the above problems.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] The present application discloses a preparation method of biocompatible membrane for biosensor, which comprises the following steps:

[0008] Step 01, main chain monomer polymerization; the main chain monomer and the initiator are mixed in a first solvent in a certain proportion, and the reaction is carried out under first reaction conditions, and then the reaction product is separated and purified to obtain a first intermediate product;

[0009] Step 02, side chain modification; the first intermediate product and the side chain modifier are mixed in a second solvent, and the reaction is carried out under second reaction conditions, and then the reaction product is separated and purified to obtain a second intermediate product;

[0010] Step 03, side chain coenzyme modification; the second intermediate product and the side chain modification coenzyme are mixed in a third solvent, and the reaction is carried out under third reaction conditions, and then the reaction product is separated and purified to obtain a coenzyme grafted polymer;

[0011] Step 04, coating and film forming; the coenzyme grafted polymer and a fourth solvent are uniformly mixed to form a film solution, and then the film solution is coated to form the biocompatible film by using a coating process.

[0012] Another aspect of the present application discloses a glucose sensor, which comprises an electrode, and a biocompatible film prepared by the method for preparing a biocompatible film for a biosensor is wrapped outside a sensing layer of the electrode.

[0013] In summary, the beneficial effects of the present application are:

[0014] The biocompatible film prepared by the main chain monomer polymerization reaction, the side chain modification reaction, the side chain coenzyme modification reaction and the coating and film forming can be applied to a biosensor, which not only can realize accurate regulation of chemical substance penetration, expand the linear range of the biosensor for chemical substance detection, make the biosensor accurately and sensitively detect the concentration of the chemical substance, but also can provide coenzyme for the biosensor, thereby increasing the stability and durability of the biosensor, and making the biosensor can be continuously detected for multiple times. BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 is an embodiment step diagram of the method for preparing a biocompatible film for a biosensor in an embodiment of the present application;

[0016] FIG. 2 is a curve diagram of the relative current detected by a glucose sensor without a biocompatible film and a glucose sensor with a biocompatible film with respect to the change of glucose concentration;

[0017] FIG. 3 is a curve diagram of the change of the current detected by a glucose sensor with a biocompatible film with respect to time when detecting the glucose concentration;

[0018] Figure 4 is a graph of current detected by a glucose sensor coated with a biocompatible membrane as a function of glucose concentration at different times. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be described in further detail below with reference to the drawings.

[0020] In one embodiment of the present application, a preparation method of a biocompatible membrane for a biosensor is provided, as shown in Figure 1, which comprises the following steps:

[0021] Step 01, main chain monomer polymerization; the main chain monomer and the initiator are mixed in a first solvent in a certain proportion and reacted under first reaction conditions, and then the reaction product is separated and purified to obtain a first intermediate product; wherein the reaction under the first reaction conditions is specifically: reacting for 6h-18h under an anaerobic environment at 30-80℃; the mass ratio of the main chain monomer to the initiator is 50-500:1.

[0022] Step 02, side chain modification; the first intermediate product and the side chain modifier are mixed in a second solvent and reacted under second reaction conditions, and then the reaction product is separated and purified to obtain a second intermediate product; wherein the reaction under the second reaction conditions is specifically: reacting for 12h-24h under an anaerobic environment at 40-90℃; the mass ratio of the first intermediate product to the side chain modifier is 0.5-2:1.

[0023] Step 03, side chain coenzyme modification; the second intermediate product and the side chain modification coenzyme are mixed in a third solvent and reacted under third reaction conditions, and then the reaction product is separated and purified to obtain a coenzyme grafted polymer; wherein the reaction under the third reaction conditions is specifically: reacting for 6h-18h under an anaerobic environment at 30-80℃; the mass ratio of the second intermediate product to the side chain modification coenzyme is 2-10:1.

[0024] Step 04, coating and film forming; the coenzyme grafted polymer and a fourth solvent are uniformly mixed to form a film solution, and then a coating process is used to coat the film solution to form a biocompatible membrane. After the formation of the biocompatible membrane, it needs to be dried by baking or standing.

[0025] It should be noted that, since the coating and film forming process is independent of the synthesis process of the cofactor grafted polymer, the cofactor grafted polymer is separated and stored by purification after being prepared, and a film solution is prepared when it is needed to be used for coating and film forming; in this way, the stability and service life of the film solution are greatly improved, the stability and service life of the same batch of products do not change significantly within one year, the consistency of the biocompatible film is ensured, and the high consistency of the prepared implantable continuous glucose detection system containing the biocompatible film is ensured.

[0026] In summary, the biocompatible film prepared by the main chain monomer polymerization reaction, the side chain modification reaction, the side chain cofactor modification reaction and the coating and film forming in sequence in the embodiment can be applied to a biosensor, and specifically wrapped on the outside of the biosensor electrode sensing layer. The biocompatible film not only can realize accurate regulation of chemical substance penetration, expand the linear range of the biosensor for chemical substance detection, i.e., limit the sensing layer chemical substance sensing concentration on the biosensor, expand the linear range of the current value collected by the biosensor and the chemical substance concentration, enable the biosensor to accurately and sensitively detect the concentration of the chemical substance, but also can provide the biosensor with cofactors, thereby increasing the stability and durability of the biosensor, and enabling the biosensor to perform continuous multiple detections.

[0027] In the embodiment, the main chain monomer is one or more of urethane monomer, vinyl alcohol monomer, vinyl pyrrolidone monomer and vinyl pyridine monomer, and is preferably vinyl pyridine monomer. The side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, bromobutyric acid ethyl ester, bromohexanoic acid and bromohexanoic acid ethyl ester, and is preferably bromohexanoic acid. The biocompatible film prepared by using the above-mentioned types of main chain monomers and side chain modifiers as raw materials has high biocompatibility.

[0028] In addition, the initiator is one or more of azobisisobutyronitrile, azobisisoheptyl nitrile and benzoyl peroxide.

[0029] In addition, the first solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the second solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; and the third solvent includes water, and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide. The first solvent, the second solvent and the third solvent can be the same type of solvent or different types of solvents.

[0030] Further, the mass concentration of the cofactor grafted polymer in the membrane solution is 15 mg / mL to 150 mg / mL, preferably 35 mg / mL to 70 mg / mL, and most preferably 60 mg / mL. In this way, a sufficient amount of cofactor can be provided for the biosensor.

[0031] In the present embodiment, the fourth solvent includes water, an organic solvent, and a buffer, which are uniformly mixed when used.

[0032] The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate, and dimethyl sulfoxide, and is preferably ethanol. The buffer is one of PBS buffer, HEPES buffer, TAPS buffer, and MES buffer. The volume fraction of the organic solvent in the total volume of the organic solvent and the buffer is 50% to 97%, and is preferably 85% to 95%. The volume fraction of the buffer in the total volume of the organic solvent and the buffer is 3% to 50%, and is preferably 5% to 15%. The pH of the buffer ranges from 5.5 to 9. The fourth solvent of this component enables the cofactor grafted polymer to be uniformly dissolved, thereby improving the film-forming effect of the membrane solution.

[0033] In addition, the coating process is one of dip coating, dot coating, draw coating, and vapor deposition, and is preferably dip coating.

[0034] When dip coating is used to form the film, the draw rate is 500 μm / s to 3000 μm / s, for example, 1000 μm / s to 3000 μm / s, or 500 μm / s to 1000 μm / s. The dip coating and the interval process can be repeated multiple times to increase the thickness of the biocompatible film, so that the biocompatible film can be uniformly stacked to 5 to 20 layers, and the thickness of the biocompatible film reaches 5 to 60 microns. When dip coating is performed multiple times, the interval time between adjacent two dip coatings is 0.5 min to 30 min, for example, 0.5 min to 3 min, or 10 min to 20 min. The standing and drying time after dip coating is 8 h to 24 h, for example, 8 h to 12 h, or 20 h to 24 h, etc. The standing temperature can be 20°C to 50°C, for example, 25°C to 45°C, or 30°C to 40°C, and is preferably 20°C to 25°C.

[0035] In addition, in step 04, the coating environment is a solvent vapor atmosphere and a 100,000-level clean constant temperature and humidity environment, thereby improving the quality of the film. The solvent vapor can be ethanol vapor. It should be noted that the preparation of the membrane solution and the process of coating the membrane solution to form a film need to be carried out in this environment.

[0036] In the present embodiment, the side chain modified coenzyme is one or more of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate, and is preferably nicotinamide adenine dinucleotide phosphate.

[0037] In another embodiment of the present application, a glucose sensor is provided, which comprises an electrode, and a biocompatible membrane prepared by the method for preparing a biocompatible membrane for a biosensor in the above embodiment is wrapped outside the sensing layer of the electrode. The glucose sensor has the advantages of high detection accuracy and good durability.

[0038] Embodiment 1

[0039] Synthesis of membrane material: first, ethylene pyridine and initiator were mixed and dissolved in N,N-dimethylformamide (DMF) at a mass ratio of 400:1, and heated in a water bath at 60°C for 10h, and the reaction product was separated by precipitation with methanol solution to obtain a first intermediate product. Then, the first intermediate product and bromoacetic acid were dissolved in N,N-dimethylformamide (DMF) at a mass ratio of 1:1, and heated in a water bath at 80°C for 18h, and the reaction product was separated by precipitation with ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product and nicotinamide adenine dinucleotide phosphate were dissolved in N,N-dimethylformamide (DMF) at a mass ratio of 10:1, and heated in a water bath at 70°C for 5h, and the reaction product was separated and purified by methanol solution, and dried to obtain the coenzyme grafted polymer.

[0040] Membrane formation on the electrode of the biosensor: first, in a 100,000-level clean room and in an environment containing saturated ethanol vapor, the coenzyme grafted polymer was dissolved in anhydrous ethanol with a volume fraction of 95% and HEPES buffer (10mM, pH=8) with a volume fraction of 5% to form a membrane solution at a concentration of 100mg / mL. Second, the membrane solution was uniformly coated on the glucose sensor containing electrochemically activated glucose dehydrogenase by dip-coating method. The dip-coating rate was 500μm / s. Then, the process of dip-coating and standing was repeated for 8 times under the condition of temperature 25°C and relative humidity 50% for 30min. Finally, the implantable glucose sensor was obtained after drying for 18h under constant temperature conditions and was used for testing.

[0041] Electrochemical test: The glucose sensor in this embodiment was immersed in a simulated human body fluid (SBF buffer) containing different concentrations of glucose, and the chronoamperometry method was used for detection, and the detection results are shown in Figure 2. When the electrode of the biosensor is covered with a biocompatible film, the relative value of the current decreases, the linear range increases, and the linear correspondence in the range of 0-30mM glucose can be met, thereby indicating that the biocompatible film can effectively limit the glucose permeation in proportion, and at the same time accurately and sensitively indicate the glucose concentration through the current. And through the comparison of the output current of the glucose sensor covered with the biocompatible film and the glucose sensor without the biocompatible film after being subjected to glucose, it can be found that the presence of the biocompatible film can make the electrode of the glucose sensor have a wider linear range, which is more suitable for the working environment of continuous blood glucose detection.

[0042] Example 2

[0043] Synthesis of film material: First, ethylene pyridine and initiator were mixed and dissolved in N,N-dimethylformamide (DMF) in a mass ratio of 400:1, and heated in a water bath at 60℃ for 10h, and the reaction product was separated by precipitation with methanol solution to obtain a first intermediate product. Then, the first intermediate product and bromoacetic acid were dissolved in N,N-dimethylformamide (DMF) in a mass ratio of 1:1, and heated in a water bath at 80℃ for 18h, and the reaction product was separated by precipitation with ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product and nicotinamide adenine dinucleotide phosphate were dissolved in N,N-dimethylformamide (DMF) in a mass ratio of 10:1, and heated in a water bath at 70℃ for 5h, and then the reaction product was separated and purified by methanol solution, and dried to obtain the cofactor grafted polymer.

[0044] Film formation on the electrode of the biosensor: First, in a 100,000-level clean room and an environment containing saturated ethanol vapor, the cofactor grafted polymer was dissolved in anhydrous ethanol with a volume fraction of 95% and HEPES buffer (10mM, pH=8) with a volume fraction of 5% to form a biocompatible film solution at a concentration of 60mg / mL. Second, the biocompatible film solution was uniformly coated on the glucose sensor containing electrochemically activated glucose dehydrogenase by dip-coating method. Among them, the pulling rate is 500μm / s. Then, it is placed for 30min in an environment with temperature of 25℃ and relative humidity of 50%, and the process of dip-coating and standing is repeated for 8 times. Finally, it is dried for 18h under constant temperature conditions to obtain an implantable glucose sensor for testing.

[0045] Electrochemical test: The glucose sensor in this example was immersed in a human simulation solution (SBF buffer) containing glucose, and chronoamperometry was used to detect the change in stability (current) over a long period of time (35 days). Figure 3 is a graph showing the change in current of the glucose sensor in Example 2 in an SBF buffer solution containing 10 mM glucose over 35 days. As can be seen, when the biosensor is covered with a biocompatible film, the current hardly changes over 35 days and can be stably maintained at the same level (change amplitude < 7%). Figure 4 shows the glucose concentration linear titration data of the glucose sensor electrode on day 1, day 7, day 14, day 20, day 30 and day 35. The glucose concentration-electrical signal of the glucose sensor has a linear relationship over 35 days, and the signal size is almost unchanged. The above electrochemical test results show that the biocompatible film can maintain the linearity, stability and sensitivity of the signal response of the glucose sensor electrode for a long time.

[0046] The above description is only a specific implementation of the present application. Based on the above teaching of the present application, those skilled in the art can make other improvements or modifications on the basis of the above examples. Those skilled in the art should understand that the above specific description is only to better explain the purpose of the present application, and the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for the preparation of a biocompatible membrane for a biosensor, characterized in that, The preparation method comprises the following steps: Step 01, main chain monomer polymerization; the main chain monomer and the initiator are mixed in a first solvent in a certain proportion, and are reacted under first reaction conditions, and then the reaction product is separated and purified to obtain a first intermediate product; Step 02, side chain modification; the first intermediate product and a side chain modifier are mixed in a second solvent, and are reacted under second reaction conditions, and then the reaction product is separated and purified to obtain a second intermediate product; Step 03, side chain coenzyme modification; the second intermediate product and a side chain modification coenzyme are mixed in a third solvent, and are reacted under third reaction conditions, and then the reaction product is separated and purified to obtain a coenzyme grafted polymer; Step 04, coating and film forming; The coenzyme grafted polymer and a fourth solvent are uniformly mixed to form a film solution, and then a coating process is used to coat the film solution to form the biocompatible film.

2. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The main chain monomer is one or more of urethane monomer, vinyl alcohol monomer, vinyl pyrrolidone monomer and vinyl pyridine monomer.

3. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The side chain modifier is one or more of dibromoacetic acid, bromobutyric acid, bromobutyric acid ethyl ester, bromohexanoic acid and bromohexanoic acid ethyl ester.

4. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The reaction under the first reaction conditions is specifically 6h-18h of reaction under an oxygen-free environment at 30-80°C.

5. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The reaction under the second reaction conditions is specifically 12h-24h of reaction under an oxygen-free environment at 40-90°C.

6. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The reaction under the third reaction conditions is specifically 6h-18h of reaction under an oxygen-free environment at 30-80°C.

7. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The initiator is one or more of azobisisobutyronitrile, azobisisoheptyl nitrile and benzoyl peroxide.

8. The method for preparing a biocompatible membrane for a biosensor according to claim 7, characterized in that, The mass ratio of the main chain monomer to the initiator is 50-500:

1.

9. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The mass ratio of the first intermediate product to the side chain modifier is 0.5-2:

1.

10. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The mass ratio of the second intermediate product to the side chain modification coenzyme is 2-10:

1.

11. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The first solvent comprises water and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; the second solvent comprises water and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; and the third solvent comprises water and one or more of ethanol, methanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide.

12. The method for preparing a biocompatible membrane for a biosensor according to claim 1, characterized in that, The mass concentration of the coenzyme grafted polymer in the film solution is 15mg / mL-150mg / mL.

13. The method of claim 1, wherein the biocompatible membrane for a biosensor is prepared by a method comprising: The fourth solvent comprises water, an organic solvent and a buffer solution; The organic solvent is one or more of ethanol, tetrahydrofuran, N,N-dimethylformamide, ethyl acetate and dimethyl sulfoxide; and the buffer solution is one of PBS buffer solution, HEPES buffer solution, TAPS buffer solution and MES buffer solution.

14. The method for preparing a biocompatible membrane for a biosensor according to claim 13, characterized in that, The volume ratio of the buffer solution in the total volume of the organic solvent and the buffer solution is 3%-50%; and the pH range of the buffer solution is 5.5-9.

15. The method of claim 1, wherein the biocompatible membrane for a biosensor is prepared by, The coating process is one of dip coating, point coating, draw coating and vapor deposition.

16. The method for preparing a biocompatible membrane for a biosensor according to claim 15, characterized in that, In step 04, the coating environment is a solvent vapor atmosphere, and the temperature and humidity are controlled to 100,000-level clean.

17. The method of claim 1, wherein the biocompatible membrane for a biosensor is prepared by, The side chain modification coenzyme is one or more of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide, and reduced nicotinamide adenine dinucleotide phosphate.

18. A glucose sensor, characterized in that, The glucose sensor includes an electrode, and a biocompatible membrane prepared by the method for preparing a biocompatible membrane for a biosensor described in claim 17 is wrapped outside a sensing layer of the electrode.

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